Phospholipids are a naturally-occurring class of compounds which have been shown to be involved in various cellular functions. For example, phospholipids are integral components of cell membranes and lung surfactants, and play a major role in intracellular and extracellular signal transduction and cellular inflammatory pathways.
Phospholipids typically are composed of a glycerol backbone which is acylated with fatty acids at the C.sub.1 and C.sub.2 positions and phosphorylated at the remaining terminus. The fatty acids may or may not be identical, and may vary in degree of saturation. In one subclass of phospholipids, termed ether phospholipids, an ether linkage with an aliphatic chain is formed at the C.sub.1 position. Phospholipids also may be esterified with "head groups" on the phosphate moiety. Some commonly occurring head groups include ethanolamine (phosphatidylethanolamine), choline (phosphatidylcholine), glycerol (phosphatidylglycerol), serine (phosphatidylserine) and inositol (phosphatidylinositol).
Various protocols for synthesizing phospholipid-saccharide conjugates have been published in the literature. The traditional approach is to combine phosphatidylethanolamine with a reducing saccharide to form an imine, which is then reduced by sodium cyanoborohydride to give a stable amino linkage (P. W. Tang et al. (1985) Biochem. Biophys. Res. Comm., 132:474-480). However, reductive amination destroys the ring structure of the reducing terminus of the saccharide, which may lead to changes in the biological activity of resultant phospholipid-saccharide conjugates. Phospholipid-saccharide conjugates have also been prepared enzymatically. For example, Phospholipase-D enzymes isolated from various organisms including Streptomyces (S. Shuto, S. Imamura, K. Fukukawa, T. Ueda (1988) Chem. Pharm. Bull., 36: 5020-5023), Actinomedura and Nocardiopsis (Y. Kokusho et al., U.S. Pat. No. 4,624,919, issued Nov. 25, 1986) have been demonstrated to catalyze the transfer of a phosphatidyl residue from phosphatidylcholines to a primary hydroxy group of the saccharide. However, this method precludes any modification of the distance between the phospholipid and the saccharide. As a result, the accessibility of the saccharide residues may be limited sterically when the phospholipid-saccharide conjugates are incorporated into liposomes.
The chemical synthesis of phospholipid-galactose conjugates has been reported (J. Haensler et al. (1991) Glycoconjugate J., 8: 116-124). The synthetic scheme involves preparing 2'-carboxyethyl-1-thiogalactoside in four steps from commercially available peracetylated galactose. The carboxylic acid on the galactoside is further refunctionalized with 1,3-diamino-2-propanol via amidation. The derivatized galactose is then coupled to the N-hydroxysuccinimide ester of N-succinyl phosphatidylethanolamine. As described in the report, the linker between the phospholipid and the galactose in the final product contains a minimum of sixteen C--C, C--O and C--N bonds. This procedure is not suitable if conjugates having a shorter linker are desired.
Phospholipids have been used extensively as components for liposomes. Liposomes are lipid vesicles which can be used to encapsulate and deliver drugs to cell tissues. Conventional liposomes, however, are rapidly cleared from the bloodstream by the reticuloendothelial system (RES). In order to improve the targeting capabilities of the liposomes, second generation liposomes have been constructed. These improved liposomes generally can be catagorized into two types: one type has an extended circulatory lifetime, the other type has the ability to target specific tissues via cellular interactions between the liposome and the target cell.
Phospholipids modified with saccharides have been utilized in preparing both types of liposomes. For example, liposomes containing G.sub.M1 have been shown to exhibit the ability to avoid RES uptake, though the mechanism of avoidance is not yet well understood. Liposomes composed of G.sub.M1 -distearoylphosphatidyl-choline-cholesterol, when compared to formulations containing combinations of phosphatidylserine, phosphatidylcholine and cholesterol, show high levels of accumulation in blood and concommitant low levels in liver and spleen (A. Gabison, D. Papahadjopoulos (1988) Proc. Natl Acad. Sci., 85:6949-6953). Ligand-receptor interactions have been used to construct liposomes targeted to specific cell tissues. For example, mannosylated phosphatidylinositols, which are extracted from the cell wall of mycobacteria, have been incorporated in liposomal systems and shown to be effective in delivering therapeutic agents to macrophages via the cell-surface mannose receptors (G. Barratt et al. (1986) Biochim. Biophys. Acta, 862:153-164). Liposomes containing p-aminophenyl mannoside were reported to enhance the transport mechanism across the blood-brain barrier (F. Umezawa, Y. Eto (1988) Biochem. & Biophys. Res. Comm., 153:1038-1044). Vesicles containing conjugates of cholesterol with 6-amino-6-deoxy-mannoside show significant ability to enhance the uptake of the vesicles by mouse peritoneal macrophages, where receptors for mannose-terminated glycoprotein are located (P.-S. Wu, G. W. Tin, J. Baldeschwieler (1981) Proc. Natl. Acad. Sci. U.S.A., 78:2033-2037). Haensler and F. Schuba (Biochim. Biophys. Acta, 946:95-105 (1988)) describe galactosylated liposomes. They report that results from studies directed toward the interaction of these liposomes with mouse peritoneal macrophages show a 4- to 5-fold increase in binding of the liposomes to the cells.
None of the liposomes heretofore described display effective levels of tissue specificity and resistance to degradation in vivo, however. Phospholipid-sugar conjugates which could be used to make cell-targeted lipsomes or which are resistant to RES uptake would be useful.